In vitro detection of anti-TB liver metabolites in early drug discovery Abstract Current algorithms for high throughput screening-based drug discovery for antimicrobial agents, including those for tuberculosis, fail to account for the possibility of active metabolites early in the drug discovery process. Compounds that would only be active after metabolism in the liver (prodrugs) are not detected in high throughput screens. Similarly, compounds that are found to be metabolically unstable by LC-MS during in vitro ADME evaluation are not progressed to in vivo evaluation on the assumption that metabolites will be inactive (type 2 compounds). Proving that assumption would require identification of the metabolite structure, synthesis and testing the metabolite directly. This is too time and labor intensive at the hit to lead stage. We will establish in vitro assays to rapidly detect the anti-tuberculosis (TB) activity of liver enzyme-derived metabolites. These TB-active metabolite assays (TAMA) should obviate the need for metabolite ID and synthesis to confirm the presence or absence of an active metabolite. We will combine liver enzyme metabolite generation systems (MGS) with rapid anti-TB assays. Enzymatic MGS includes liver microsomes, S9 fractions, recombinant CYP450 enzymes and combinations of these systems and can be used in a one-pot assay. Preliminary data suggests enzymatic MGS are compatible with M. tuberculosis growth and rapid viability assessment by fluorometric determination of Alamar Blue reduction or luminescent intracellular ATP measurement. HepaRG and MCL-5 cell supernatants will also be assessed as MGS. To distinguish active metabolites of active parent compounds (type 3) from metabolically stable, active parent compounds (type 1) concurrent LC-MS analysis of parent compound stability will be performed and analyzed together with the TAMA data. An optimized TAMA will be used in a HTS of 100K compounds to identify prodrugs and potential type 3 compounds. Determination of stability of all hits by LC-MS and subsequent data analysis will differentiate type 3 from type 1 compounds. Selected metabolites will be identified by LC-MS/MS and NMR, synthesized and evaluated directly against M. tuberculosis as proof of concept. The MGS can increase compound library diversity and give consideration to revisiting previously screened libraries. The ability to detect active metabolites by this method early in the drug discovery process will allow for the further progression of some active (type 3) compounds with poor metabolic stability that otherwise would be deprioritized using only LC-MS based stability assays.